1. Field of the Invention
The present invention in the fields of molecular biology, immunology and medicine relates to chimeric nucleic acids encoding fusion proteins and their use as vaccines to enhance immune responses, primarily cytotoxic T lymphocyte (CTL) responses to specific antigens such as tumor antigens. The fusion proteins comprise an antigenic polypeptide fused to a protein that promotes processing via the MHC class I pathway and/or promotes development or activity of antigen presenting cells (APCs), primarily dendritic cells (DCs). Preparation of the foregoing nucleic acid constructs as naked DNA plasmids, self-replicating RNA replicons and suicidal DNA-based viral RNA replicons confer various advantages on these molecular vaccines.
2. Description of the Background Art
Antigen-specific cancer immunotherapy has emerged as a promising approach because it is capable of engendering specific immunity against neoplastic cells while sparing normal cells. Increasing evidence suggests that professional antigen-presenting cells (APCs), particularly dendritic cells (DCs), are central players in this process. An effective vaccine strategy includes targeting the tumor antigen to professional APCs that in turn activate antigen-specific T cells (for review, see (Chen, CH et al., J Biomed Sci. 5:231-52, 1998.).
Recently, DNA vaccines have become attractive as an approach for generating antigen-specific immunotherapy (for review, see (Robinson, H L Vaccine 15:785-778, 1997; Robinson, H L et al., Semin Immunol. 9:271-83, 1997; Pardoll, D M et al., Immunity. 3:165-9, 1995; Donnelly, J J et al., Annu Rev Immunol. 15: 617-48, 1997). The advantages of naked DNA include purity, ease of preparation and stability. In addition, DNA-based vaccines can be prepared inexpensively and rapidly in large-scale. Furthermore, multiple DNA vaccines can be administered simultaneously. However, naked DNA vaccines raise concerns such as potential integration into the host genome and cell transformation. Because they do not have the intrinsic ability to amplify in vivo as do viral vaccines, DNA vaccines may be more limited in their potency.
The present inventors conceived that a directing a DNA vaccine encoding an antigen (in the form of a fusion protein) to cells which activate immune responses, such as DCs, would enhance the vaccine's potency. Others demonstrated that linking DNA encoding the cytokine GM-CSF gene to DNA encoding an HIV or hepatitis C antigen enhanced the potency of DNA vaccines (Lee, A H et al., Vaccine 17: 473-9, 1999; Lee, S W et al., J Virol. 72: 8430- 6, 1998). The chigmeric GM-CSF/antigen is believed to act as an immunostimulatory signal to DCs, inducing their differentiation from an immature form (Banchereau, J et al., Nature 392: 245-52, 1998). Since DCs and their precursors express high levels of GM-CSF receptors, the chimeric GM-CSF/antigen should target and concentrate the linked antigen to the DCs and further improve the vaccine's potency.
Use of self-replicating RNA vaccines (RNA replicons) has also been identified as an important strategy in nucleic acid vaccine development. RNA replicon vaccines may be derived from alphavirus vectors, such as Sindbis virus (Hariharan, M J et al., 1998. J Virol 72:950-8.), Semliki Forest virus (Berglund, P M et al., 1997. AIDS Res Hum Retroviruses 13:1487-95; Ying, H T et al., 1999. Nat Med 5:823-7) or Venezuelan equine encephalitis virus (Pushko, P M et al., 1997. Virology 239:389-401). These self-replicating and self-limiting vaccines may be administered as either (1) RNA or (2) DNA which is then transcribed into RNA replicons in cells transfected in vitro or in vivo (Berglund, P C et al., 1998. Nat Biotechnol 16:562-5; Leitner, W W et al., 2000. Cancer Res 60:51-5).
Self-replicating RNA infects a diverse range of cell types and allows the expression of a linked antigen of interest at high levels (Huang, H V 1996. Curr Opin Biotechnol 7:531-5) Because viral replication is toxic to infected host cells, such self-replicating RNA preparations eventually causes lysis of the transfected cells (Frolov, I et al., 1996. J Virol 70:1182-90). These vectors cannot integrate into the host genome, and therefore do not raise concerns of associated with naked DNA vaccines. This is particularly important for vaccine development where target proteins are potentially oncogenic, such as human papillomavirus (HPV) E6 and E7 proteins.
The present inventors and their colleagues recently demonstrated that linkage of HPV-16 E7 antigen to Mtb heat shock protein 70 (Hsp70) leads to the enhancement of DNA vaccine potency (Chen, CH et al., 2000. Cancer Research 60:1035-1042). (See also co-pending patent applications U.S. Ser. No. 09/501,097, filed 9 Feb. 2000; and U.S. Ser. No. 099/421,608, filed 20 Oct. 1999, all of which are incorporated by reference in their entirety.) Immunization with HSP complexes isolated from tumor or virus-infected cells induced potent anti-tumor immunity (Janetzki, S et al., 1998. J Immunother 21:269-76) or antiviral immunity (Heikema, A E et al, Immunol Lett 57:69-74). In addition, immunogenic HSP-peptide complexes dould be reconstituted in vitro by mixing the peptides with HSPs (Ciupitu, AM et al., 1998. J Exp Med 187:685-91). Furthermore, HSP-based protein vaccines have been created by fusing antigens to HSPs (Suzue, K et al., 1996. J Immunol 156:873-9). The results of these investigations point to HSPs a attractive candidates for use in immunotherapy. However, prior to the present inventors' work, HSP vaccines were all peptide/protein-based vaccines or, in more recent cases, were in the form of naked DNA. To date, there have been no reports of HSPs incorporated into self-replicating RNA vaccines.
Another molecule that stimulates growth of DC precursors and can help in generating large numbers of DCs in vivo is Flt3-ligand (“FL”) (Maraskovsky, E et al., J Exp Med 184: 1953-62, 1996, Shurin, M R et al., Cell Lmmunol. 179: 174-84, 1997). FL has emerged as an important molecule in the development of tumor vaccines that augment numbers and action of DCs in vivo. Flt3, a murine tyrosine kinase receptor, first described in 1991 (Rosnet, O et al., Oncogene. 6: 1641-50, 1991), was found to be a member of the type III receptor kinase family which includes -kit and c-fms (for review, see (Lyman, S D Curr Opin Hematol. 5:192-6, 1998). In hematopoietic tissues, the Flt3expression is restricted to the CD34+ progenitor population. Flt3 has been used to identify and subsequently clone the corresponding ligand, Flt3-ligand or “FL” (Lyman, S D et al., Cell. 75: 1157-67, 1993; Hannum, C et al., Nature. 368: 643-8, 1994).
The predominant form of FL is synthesized as a transmembrane protein from which the soluble form is believed to be generated by proteolytic cleavage. The soluble form of FL (the extracellular domain or “ECD”) is functionally similar to intact FL (Lyman, S D et al., Cell. 75: 1157-67, 1993). These proteins function by binding to and activating unique tyrosine kinase receptors. Expression of the Flt3 receptor is primarily restricted, among hematopoietic cells, to the most primitive progenitor cells, including DC precursors. The soluble ECD of FL induced strong anti-tumor effects against several murine model tumors including fibrosarcoma (Lynch, D H et al., Nat Med. 3: 625-31, 1997), breast cancer (Chen, K et al Cancer Res. 57: 3511-6, 1997; Braun, S E et al., Hum Gene Ther. 10: 2141-51, 1999), liver cancer (Peron, J M et al., J Immunol. 161: 6164-70, 1998), lung cancer (Chakravarty, P K et al., Cancer Res. 59: 6028-32, 1999), melanoma and lymphoma (Esche, C et al., Cancer Res. 58: 380-3, 1998).
There is a need in the art for improved molecular vaccines, such as nucleic acid vaccines, that combine potency and safety. The present invention helps meet this need by its design of novel fusion or chimeric polypeptides and nucleic acids coding therefor, that link the antigen with specialized polypeptides that promote antigen presentation by various mechanisms and that exploit delivery of these constructs by various nucleic acid vectors.